Abstract

Morphological changes and complex developmental processes inside vertebrate embryos are difficult to observe noninvasively with millimeter-penetration and sub-micrometer-resolution at the same time. By using higher harmonic generation, including second and third harmonics, as the microscopic contrast mechanism, optical noninvasiveness can be achieved due to the virtual-level-transition characteristic. The intrinsic nonlinearity of harmonic generations provides optical sectioning capability while the selected 1230-nm near-infrared light source provides the deep-enetration ability. The complicated development within a ~1.5-mm thick zebrafish (Danio rerio) embryo from initial cell proliferation, gastrulation, to tissue formation can all be observed clearly in vivo without any treatment on the live specimen.

© 2003 Optical Society of America

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  1. G. Peleg, A. Lewis, M. Linial, and L. M. Loew, �??Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites,�?? Proc. Natl. Acad. Sci. 96, 6700-6704 (1999).
    [CrossRef] [PubMed]
  2. S.-W. Chu, I-S. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, �??Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy,�?? J. Microscopy 208, 190-200 (2002).
    [CrossRef]
  3. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, �??Three dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,�?? Biophys. J. 82, 493-508 (2002).
    [CrossRef]
  4. M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, �??3D microscopy of transparent objects using third-harmonic generation,�?? J. Microsc. 191, 266-274 (1998).
    [CrossRef] [PubMed]
  5. D. Yelin and Y. Silberberg, �??Laser scanning third-harmonic-generation microscopy in biology,�?? Opt. Express 5, 169-175 (1999) <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-5-8-169">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-5-8-169</a>
    [CrossRef] [PubMed]
  6. L. Canioni, S. Rivet, L. Sarger, R. Barille, P. Vacher, and P. Voisin, �??Imaging of Ca2+ intracellular dynamics with a third-harmonic generation microscope,�?? Opt. Lett. 26, 515-517 (2001).
    [CrossRef]
  7. A. Y. Louie, M. M. Hüber, E. T. Ahrens, U. Rothbächer, R. Moats, R. E. Jacobs, S. E. Fraser, and T. J. Meade, �??In vivo visualization of gene expression using magnetic resonance imaging,�?? Nat. Biotech. 18, 321-325 (2000).
    [CrossRef]
  8. F. S. Foster, C. J. Pavlin, K. A. Harasiewicz, D.A. Christopher, and D. H. Turnbull, �??Advances in ultrasound biomicroscopy,�?? Ultrasound in Med. Biol. 26, 1�??27 (2000).
    [CrossRef]
  9. S. A. Boppart, G. J. Tearney, B. E. Bouma, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, �??Noninvasive assessment of the developing Xenopus cardiovascular system using optical coherence tomography,�?? Proc. Natl. Acad. Sci. 94, 4256�??4261 (1997).
    [CrossRef] [PubMed]
  10. T. M. Yelbuz, M. A. Choma, L. Thrane, M. L. Kirby, and J. A. Izatt, �??Optical coherence tomography: A new high-resolution imaging technology to study cardiac development in chick embryos,�?? Circulation 106, 2771-2774 (2002).
    [CrossRef] [PubMed]
  11. C. Palmes-Saloma and C. Saloma, �??Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH,�?? J. Struct. Bio. 131, 56�??66 (2000).
    [CrossRef]
  12. J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, �??Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,�?? Nat. Biotech. 17, 763-767 (1999).
    [CrossRef]
  13. C. L. Phillips, L. J. Arend, A. J. Filson, D. J. Kojetin, J. L. Clendenon, S. Fang, and K. W. Dunn, �??Three dimensional imaging of embryonic mouse kidney by two-photon microscopy,�?? Am. J. Pathol. 158, 49-55 (2001).
    [CrossRef] [PubMed]
  14. R. R. Anderson and J. A. Parish, �??The optics of human skin,�?? J. Invest. Dermat. 77, 13-19 (1981).
    [CrossRef]
  15. B. E. Bouma, G. J. Tearney, I. P. Bilinsky, B. Golubovic, and J. G. Fujimoto, �??Self-phase-modulated Kerrlens mode-locked Cr:forsterite laser source for optical coherence tomography,�?? Opt. Lett. 21, 1839 (1996).
    [CrossRef] [PubMed]
  16. S.-W. Chu, I-H. Chen, T.-M. Liu, P. C. Cheng, C.-K. Sun, and B.-L. Lin, �??Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser,�?? Opt. Lett. 26, 1909-1911 (2001).
    [CrossRef]
  17. T.-M. Liu, S.-W. Chu, C.-K. Sun, B.-L. Lin, P. C. Cheng, and I. Johnson, �??Multi-photon scanning microscopy using a femtosecond Cr:forsterite laser,�?? Scanning 23, 249-254 (2001).
    [CrossRef] [PubMed]
  18. A. Seas, V. Petri�?evi�?, and R.R. Alfano, �??Generation of sub-100-fs pulses from a CW mode-locked chromium-doped forsterite laser,�?? Opt. Lett. 17, 937-939 (1992).
    [CrossRef] [PubMed]
  19. C. B. Kimmel, W. W. Ballard, S. R. Kimmel, B. Ullmann, and T. F. Schilling, �??Stages of embryonic development of the zebrafish,�?? Dev. Dynam. 203, 253-310 (1995).
    [CrossRef]
  20. J. M. Schins, T. Schrama, J. Squier, G. J. Brakenhoff, and M. Müller, �??Determination of material properties by use of third-harmonic generation microscopy,�?? J. Opt. Soc. Am. B 19, 1627-1634 (2002).
    [CrossRef]
  21. K. König, P.T.C. So, W.W. Mantulin, and E. Gratton, �??Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes,�?? Opt. Lett. 22, 135-136 (1997).
    [CrossRef] [PubMed]
  22. A. Schönle and S. W. Hell, �??Heating by absorption in the focus of an objective lens,�?? Opt. Lett. 23, 325-327 (1998)
    [CrossRef]
  23. I-H. Chen, S.-W. Chu, C.-K. Sun, P. C. Cheng, and B.-L. Lin, �??Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,�?? Opt. Quantum. Electron 34, 1251-1266 (2002).
    [CrossRef]
  24. S.-W. Chu, T.-M. Liu, C.-K. Sun, C.-Y. Lin, and H.-J. Tsai, �??Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti:sapphire laser,�?? Optics Express 11, 933-938 (2003) <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-8-933">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-8-933</a>
    [CrossRef] [PubMed]

Am. J. Pathol.

C. L. Phillips, L. J. Arend, A. J. Filson, D. J. Kojetin, J. L. Clendenon, S. Fang, and K. W. Dunn, �??Three dimensional imaging of embryonic mouse kidney by two-photon microscopy,�?? Am. J. Pathol. 158, 49-55 (2001).
[CrossRef] [PubMed]

Biophys. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, �??Three dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,�?? Biophys. J. 82, 493-508 (2002).
[CrossRef]

Circulation

T. M. Yelbuz, M. A. Choma, L. Thrane, M. L. Kirby, and J. A. Izatt, �??Optical coherence tomography: A new high-resolution imaging technology to study cardiac development in chick embryos,�?? Circulation 106, 2771-2774 (2002).
[CrossRef] [PubMed]

Dev. Dynam.

C. B. Kimmel, W. W. Ballard, S. R. Kimmel, B. Ullmann, and T. F. Schilling, �??Stages of embryonic development of the zebrafish,�?? Dev. Dynam. 203, 253-310 (1995).
[CrossRef]

J. Invest. Dermat.

R. R. Anderson and J. A. Parish, �??The optics of human skin,�?? J. Invest. Dermat. 77, 13-19 (1981).
[CrossRef]

J. Microsc.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, �??3D microscopy of transparent objects using third-harmonic generation,�?? J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

J. Microscopy

S.-W. Chu, I-S. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, �??Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy,�?? J. Microscopy 208, 190-200 (2002).
[CrossRef]

J. Opt. Soc. Am. B

J. Struct. Bio.

C. Palmes-Saloma and C. Saloma, �??Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH,�?? J. Struct. Bio. 131, 56�??66 (2000).
[CrossRef]

Nat. Biotech.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, �??Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,�?? Nat. Biotech. 17, 763-767 (1999).
[CrossRef]

A. Y. Louie, M. M. Hüber, E. T. Ahrens, U. Rothbächer, R. Moats, R. E. Jacobs, S. E. Fraser, and T. J. Meade, �??In vivo visualization of gene expression using magnetic resonance imaging,�?? Nat. Biotech. 18, 321-325 (2000).
[CrossRef]

Opt. Express

Opt. Lett.

Opt. Quantum. Electron

I-H. Chen, S.-W. Chu, C.-K. Sun, P. C. Cheng, and B.-L. Lin, �??Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,�?? Opt. Quantum. Electron 34, 1251-1266 (2002).
[CrossRef]

Optics Express

S.-W. Chu, T.-M. Liu, C.-K. Sun, C.-Y. Lin, and H.-J. Tsai, �??Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti:sapphire laser,�?? Optics Express 11, 933-938 (2003) <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-8-933">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-8-933</a>
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci.

S. A. Boppart, G. J. Tearney, B. E. Bouma, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, �??Noninvasive assessment of the developing Xenopus cardiovascular system using optical coherence tomography,�?? Proc. Natl. Acad. Sci. 94, 4256�??4261 (1997).
[CrossRef] [PubMed]

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, �??Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites,�?? Proc. Natl. Acad. Sci. 96, 6700-6704 (1999).
[CrossRef] [PubMed]

Scanning

T.-M. Liu, S.-W. Chu, C.-K. Sun, B.-L. Lin, P. C. Cheng, and I. Johnson, �??Multi-photon scanning microscopy using a femtosecond Cr:forsterite laser,�?? Scanning 23, 249-254 (2001).
[CrossRef] [PubMed]

Ultrasound in Med. Biol.

F. S. Foster, C. J. Pavlin, K. A. Harasiewicz, D.A. Christopher, and D. H. Turnbull, �??Advances in ultrasound biomicroscopy,�?? Ultrasound in Med. Biol. 26, 1�??27 (2000).
[CrossRef]

Supplementary Material (4)

» Media 1: MOV (730 KB)     
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» Media 3: MOV (738 KB)     
» Media 4: MOV (280 KB)     

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Figures (3)

Fig. 1.
Fig. 1.

Mitosis processes inside a live zebrafish embryo in vivo monitored with HOM. (a) An optical section of the embryo at the dome stage (4-hpf). The imaging depth is about 400-µm from the chorion surface. THG (shown in blue throughout this paper) picks up all interfaces including external yolk syncytial layers, cell membranes, and nuclear membranes while SHG (shown in green throughout this paper) shows the microtubule-formed spindle biconical array (indicated by the arrow). (b) (730-kB) Time series of the mitosis process in the embryonic blastoderm at 1-k cell stage (2.5-hpf). The cell nuclear membrane (arrowhead) and centrosomes (arrows) can be visualized through THG and SHG respectively. (c) (434 kB) Time series of the mitosis process in the embryonic blastoderm at shield-stage (6-hpf). Scale bar: 20-µm.

Fig. 2:
Fig. 2:

In vivo HOM sectioning inside a live zebrafish embryo at the 2-somite stage. (a) A sectioning showing the whole embryo at a depth of 700-µm from the chorion surface (ventral view). PL: polster; TB: tail bud. (b) THG image of the chorion surface, showing the <1-µm diameter granular canals and demonstrating the sub-µm resolution. (c) (738 kB) Depth-resolved optical section series at depths from 300-µm to 1400-µm inside the embryo. Scale bar: 100-µm except for B: 10-µm.

Fig. 3:
Fig. 3:

In vivo HOM sectioning inside a live zebrafish larva at the 20-somite stage. (a) An optical section at the center of the larva showing the segments inside the vacuolated notochord and the distribution of somites alongside the notochord. (b) The enlarged view inside a somite showing individual muscle fiber and the sarcomeres on it through SHG as well as the interface between somites through THG. (c) (281 kB) Depth resolved optical series showing that we can visualize through a whole zebrafish larva with HOM. Scale bar: 20-µm.

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